MiR-452 Regulates C2C12 Myoblast Proliferation and Differentiation via Targeting ANGPT1

microRNAs are a kind of endogenous, non-coding, single-strand small RNA. They have been reported as an important regulatory factor in skeletal myogenesis. In this study, miR-452 was selected from RNA high-throughput sequencing data to explore its regulatory role in myogenesis. Functionally, miR-452 overexpression could promote C2C12 myoblast proliferation while inhibiting myogenic differentiation. On the contrary, inhibition of miR-452 could suppress C2C12 myoblast proliferation but accelerate myogenic differentiation. Bioinformatics analysis and dual luciferase report assays showed that Angiopoietin 1 (ANGPT1), RB1, and CACNB4 were the potential target genes of miR-452. To further confirm the target relationship between ANGPT1, RB1, and CACNB4 with miR-452, the mRNA level and protein level of these genes were detected by using RT-qPCR and Western blot, respectively. Result analysis indicated that ANGPT1 was a target gene of miR-452. In addition, knockdown of ANGPT1 could obviously promote C2C12 myoblast proliferation but block their differentiation. In summary, these results demonstrated that miR-452 promoted C2C12 myoblast proliferation and inhibited their differentiation via targeting ANGPT1.


INTRODUCTION
Skeletal muscle development is an orchestral process. During the embryonic stage, myogenic progenitor cells undergo directional differentiation to form myoblasts, and after further proliferation, differentiation, and fusion, which eventually form multinuclear myotubes. The development process is regulated by myogenic regulatory factors (MRFs) and members of the myocyte enhancer factor 2 (MEF2) family, as well as by some non-coding RNAs likely microRNAs (miRNAs) (Yusuf and Brand-Saberi, 2012).
Previous research has shown that miR-452 plays different regulatory roles in various tumors. In hepatocellular carcinoma cells and a breast cancer cell line (MCF-7), miR-452 acts as a tumor-positive miRNA. In hepatocellular carcinoma cells, the expression level of miR-452 is upregulated, then promoting migration (Zheng et al., 2014) and stem-like traits by targeting Sox7 (Zheng et al., 2016). In human breast cancer cell line (MCF-7), miR-452 was downregulated and modulated chemosensitivity of breast cancer cells to Adriamycin (ADR) (Hu et al., 2014). In addition, miR-452 also acts as a tumor-negative miRNA. For example, in non-small-cell lung cancer (NSCLC), miR-452 is downregulated and promotes the invasive capability of NSCLC cells by regulating BMI1 (He et al., 2015). In

Name
Primer name Primer sequence (5 -3 )  (Goto et al., 2016). miR-452 was also involved in human osteosarcoma, which suppressed cell proliferation, promoted cell apoptosis, and led to BMI1 protein level decline . Moreover, miR-452 had been identified from the skeletal muscle of goat (Wang et al., 2014) and sheep (Zhao et al., 2016) by high-throughput sequencing. However, the function and target genes of miR-452 during C2C12 cell proliferation and differentiation are unknown. In the present study, we found that miR-452 could promote C2C12 cell proliferation and impede their differentiation by targeting Angiopoietin 1 (ANGPT1). The current studies might offer an avenue for exploring the regulation of miR-452 in myogenesis.

Plasmid Construction
Based on TargetScan and DAVID bioinformatics software analysis, the target gene ANGPT1 was selected. The wild-type 3'-UTR and the mutant 3 -UTR (the seed region target sequence of miR-452 was changed from GCAAACA to GGTTACA by using overlap PCR) of ANGPT1 were cloned into the psiCheck2 vector between the XhoI and NotI site. Moreover, the precursor sequence of miR-452 was also cloned into pcDNA3.1(+) vector with the HindIII and BamHI site. The primer sequences for purpose fragment were listed in Table 1.
a Stem-loop RT-miR-452 was used to reverse transcription of miR-452.

Cell Transfection
In order to explore the effect of miR-452 and its target gene on skeletal myogenesis, pcDNA3.1(+)-miR-452, miR-452 mimics (GenePharma, China), miR-452 inhibitor, and siRNA ( Table 2) were transfected into C2C12 myoblasts with Lipofectamine 2000 (Invitrogen), respectively. For proliferation experiments, cell transfection was performed when the cell density is up to 40∼50% with the Opti-MEM medium. After 6 h of transfection, the medium was changed into fresh growth medium (GM) for 24 and 48 h. In the differentiation experiments, transfection was performed when the cell density is up to70-80%. After 6 h of transfection, the cell medium needs to be replaced by DM.

Real-Time Quantitative PCR
Total RNA was extracted from C2C12 myoblasts using TRIzol reagent (TakaRa, Japan). A total of 1 µg RNA was reverse transcribed into cDNA by using PrimeScript RT Kit (Takara). For RNA quantification, SYBR Mix (Takara) was performed using the ABI Step One Plus real-time PCR system (Applied Biosystems) following the manufacturer's instructions. Real-time quantitative PCR (RT-qPCR) parameters were as follows: 30 s at 95 • C, 40 cycles of 95 • C for 10 s, and 60 • C for 1 min, then Melt Curve Stage. The results were normalized by the 18S and β-actin expressions through Ct 2 − Ct value. The primers for RT-qPCR were listed in Table 3.

Cell Proliferation Assay
The proliferation state of C2C12 myoblasts was detected by Cell Counting Kit (CCK)-8 and 5-ethynyl-2-deoxyuridine (EdU) in 24-well plate. First, the cultured C2C12 cells in GM were collected 24 and 48 h after transfection of pcDNA3.1(+)-miR-452, pcDNA3.1(+), miR-452 mimics, negative control (NC), miR-452 inhibitors, and anti-negative control (anti-NC). After being washed with PBS, these cells were incubated with CCK-8 reagent (EnoGene, China) for 1 h at 37 • C. Then, all treatment group cells were detected by 450 nm absorbance using microplate reader (Thermo Fisher Scientific, United States). To detect the S phase cell number, the EdU reagent (Ribobio, China) was used to perform this. Briefly, the C2C12 cells were transfected, which were cultured in GM for 24 h with medium containing 10 µM EdU before immunostaining. Images were collected using a fluorescence microscope (Nikon, Japan). ImageJ software was used to calculate the number of EdUpositive cells and Hoechst-stained cells; the ratio of EdU-positive cells was calculated with (EdU-positive cells/Hoechst-stained cells) × 100%.

Dual-Luciferase Reporter Assay
The wild-type or mutant 3 -UTR dual-luciferase reporter vector and the miR-452 mimics or the NC were co-transfected into Frontiers in Genetics | www.frontiersin.org the HEK293T cells with Lipofectamine 2000. After transfection for 48 h, the luciferase activity analysis was performed by dualluciferase reporter assay kit. Cells were cracked with 100 µl cell lysis buffer (CLB) per well, and then these plates of cells were collected, after that, 20 µl of the cell lysis supernatant was moved to 96-well plates in triplicate containing 30 µl luciferase reaction reagent per well for firefly luciferase activities, then 30 µl luciferase reaction reagent II was added into the 96-well plates per well for the Renilla luciferase activity assay. The Renilla luciferase activity was normalized to the firefly luciferase activity.

Statistical Analysis
All experiments were performed for three replicates. All experiment data in this study were presented as mean ± standard error (SE). One-way analysis of variance (ANOVA) and T-test were used for statistical analysis with software SPSS21. The value Frontiers in Genetics | www.frontiersin.org of P < 0.05 is considered significantly different, and P ≤ 0.01 is considered extremely significantly different.

MiR-452 Accelerates C2C12 Myoblast Proliferation
To verify the function of miR-452 for C2C12 myoblast proliferation. After function gain or loss of miR-452 in C2C12, we explored the mRNA expression level of Pax7 and CDK1 by the RT-qPCR analysis. The results indicated that overexpression of miR-452 promoted the expression of Pax7 and CDK1, whereas inhibition of miR-452 depressed the expression of Pax7 and CDK1 (Figures 1A-C). Meanwhile, we also detected the protein level of Pax7 and CDK1 by Western blot analysis. The results showed that the protein expression trend of Pax7 and CDK1 was in accordance with the trend of mRNA expression level (Figures 2A,B). Furthermore, we also used EdU and CCK-8 proliferation assay kit to detect the C2C12 myoblast proliferation index, and the results indicated that overexpression of miR-452 promoted C2C12 myoblast proliferation after CCK-8 assay ( Figure 3A) and increased the proportion of EdU-positive cells ( Figure 3B). However, inhibition of miR-452 presented an opposite tendency. Taken together, aforementioned results demonstrated that miR-452 promoted C2C12 myoblast proliferation.

MiR-452 Inhibits C2C12 Myoblast Differentiation
After C2C12 myoblast confluence, the growth medium was replaced by differentiation medium to induce myoblast differentiation. We detected miR-452 expression level during C2C12 myoblast differentiation stage on days 1, 3, 5, and 7. The results showed that miR-452 expression levels presented a decreased trend (Figures 4A,B). Obviously, the expression level of miR-452 was higher at the early stage of C2C12 myoblast differentiation than in the other differentiation period. So, we speculated that miR-452 might possess a function for determining C2C12 myoblast differentiation. To further elucidate the function of miR-452 for C2C12 myoblast differentiation, the myogenic marker genes like Myf5, MyoG, and Mef2c mRNA and protein expression levels were detected.
The results show that overexpression of miR-452 could reduce these marker genes mRNA (Figures 4C,E) and protein level (Figures 5A,C). On the contrary, inhibition of endogenous miR-452 could enhance the mRNA ( Figure 4D) and protein expression level (Figure 5B) of these myogenic marker genes. All these results demonstrated that miR-452 could impede C2C12 myoblast myogenic differentiation.

MiR-452 Directly Targets 3 -UTR of ANGPT1
Depending on the regulatory mechanism of miRNAs, we found that sheep gene ANGPT1 3 -UTR contained a miR-452 seed region binding site (Figures 6A,B) through bioinformatics analysis. To further valuate the target relationship between miR-452 and these genes, we cloned and inserted the wild-type or mutant 3 UTR sequence of these genes into psi-Check2 vector ( Figure 6C). The wild-type reporter vector was co-transfected with miR-452 mimic or NC into HEK293T cells. The results indicated that the ratio of Renilla/Firefly decreased significantly in the miR-452 mimic group. Meanwhile, the mutant-type reporter vector was co-transfected with miR-452 mimic or NC into HEK293 cell. However, there is no significant luciferase activity change in miR-452 mimic group compared to the control group (Figures 6C,F,G). Moreover, overexpression of miR-452 reduced the expression level of ANGPT1 mRNA and protein level in C2C12 myoblasts (Figures 6D,E). But, inhibition of endogenous miR-452 induced the expression level of ANGPT1 mRNA and protein levels in C2C12 myoblasts (Figures 6D,E). Therefore, all these results demonstrated that ANGPT1 is a direct target gene of miR-452.

Knockdown of ANGPT1 Promotes Myoblast Proliferation and Inhibits Myogenic Differentiation
The regulatory role of ANGPT1 on the proliferation and differentiation of C2C12 myoblasts is unclear. To verify the function of ANGPT1 for C2C12 myoblast proliferation and differentiation, small-interfering RNA of ANGPT1 was used to knockdown its expression level. The results indicated that the mRNA and protein expression levels of ANGPT1 were significantly reduced ( Figure 7A). After loss of ANGPT1, during C2C12 myoblast proliferation stage, the Pax7 and CDK1 protein expression levels were upregulated ( Figure 7B). However, during  (F) Dual-luciferase activity assay of the WT or MUT 3 -UTR of CACNB4. miR-452 mimics or NC were co-transfected with the wild-type or mutant 3 -UTR luciferase reporters of CACNB4 in HEK293T cells and CACNB4 protein expression after transfection with miR-452 mimics, NC, miR-452 inhibitors, and anti-negative control (anti-NC) in C2C12 myoblast differentiation in DM on D5. (G) Dual-luciferase activity assay of the WT or MUT 3 -UTR of RB1. miR-452 mimics or NC were co-transfected with the WT or MUT 3 -UTR luciferase reporters of RB1 in HEK293T cells and RB1 mRNA expression after transfection with miR-452 mimics, NC, miR-452 inhibitors, anti-NC, pcDNA3.1(+)-miR-452, and pcDNA3.1(+) in C2C12 myoblast differentiation. Data are represented as M ± SEM. **P < 0.01, *P < 0.05. The results are shown as the mean ± SEM. *P < 0.05; **P < 0.01. In this experiment, paired two-tailed t-test was performed to calculate p-value, unpaired two-tailed t-test was performed to calculate p-value.
C2C12 myoblast differentiation stage, the MyoG, Myf5, and Mef2c protein expression level were downregulated ( Figure 7C). In addition, the C2C12 myoblast proliferation status was detected by using CCK-8 reagent. The results revealed that knockdown of ANGPT1 promoted myoblast proliferation (Figures 7D,E). Meanwhile, the number of EdU-positive cells was also detected by using EdU reagent. The results showed that the proportion of EdU-positive cells increased after transfection with si-ANGPT1 (Figures 7D,E). Altogether, these results indicated that knockdown of ANGPT1 could significantly accelerate C2C12 myoblast proliferation but inhibit myogenic differentiation.

DISCUSSION
Skeletal muscle myogenesis is a complex process, which has been demonstrated to be influenced by many factors likely MRFs and MEF2. Besides these regulatory factors, some non-coding RNA has been reported to be involved in myogenesis, such as miRNAs. In this study, we found that miR-452 could promote C2C12 myoblast proliferation and impede their differentiation via targeting ANGPT1.
At the molecular level, during C2C12 myoblast proliferation stage, the mRNA and protein expression levels of Pax7 and CDK1 were detected at 24 and 48 h. Both genes are involved in myoblast proliferation. Pax7 had been reported to be involved in myogenic satellite cell maintenance and regenerative capacity (Padilla-Benavides et al., 2015). CDK1 had been demonstrated to affect Myod half-life and myogenic activity (Kitzmann et al., 1999). Our current results showed that overexpression of miR-452 significantly increased the expression level of these genes. But inhibition of miR-452 got an opposite result. During C2C12 myoblast differentiation stage, the MRFs Myf5, MyoG, and Mef2c mRNA and protein expression levels were detected on days 1, 3, 5, and 7. The results had shown that overexpression of miR-452 significantly reduced the mRNA and protein expression of these genes and vice versa.
In mammals, miRNAs affect the stability of mRNA or impede the protein translation process by targeting the 3 UTR of mRNA (Stark et al., 2005). Through Targets can, DAVID software analysis, and dual-luciferase reporter assay, we found thatANGPT1 was the target gene of miR-452. ANGPT1 encodes secretory glycoproteins, which belongs to the angiopoietin family. All angiopoietin activates endothelial specific tyrosine kinase receptor (Tie-2) through phosphorylation, thereby playing an important role in vascular development and angiogenesis (Suri et al., 1996;Yancopoulos et al., 2000). In particular, ANGPT1 can induce proliferation of endothelial cells (Abdelmalak et al., 2008). Moreover, ANGPT1 had been demonstrated to enhance skeletal muscle regeneration in mice by inducing blood vessel formation (Mofarrahi et al., 2015). In this research, we found that loss of ANGPT1 could promote C2C12 myoblast proliferation and inhibit myogenic differentiation.
In summary, the results of the current study indicated that miR-452 promoted C2C12 myoblast proliferation and inhibited their myogenic differentiation. And loss of ANGPT1 presented a similar result, which are consistent with after overexpression of miR-452. Combining with dual-luciferase reporter assay and a series of evidence suggested that ANGPT1 was a target gene of miR-452 in C2C12 myoblasts.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.